Method and compositions for enhancing immune response and for the production of in vitro mabs

ABSTRACT

The methods and compositions of the present invention are directed to enhancing an immune response and increasing vaccine efficacy through the simultaneous or sequential targeting of specific immune system components. More particularly, specific immune components, such as macrophages, dendritic cells, B cells and T cells, are individually activated by component-specific immunostimulating agents. One such component-specific immunostimulating agent is an antigen-specific, species-specific monoclonal antibody. The invention is also directed to a method for the in vitro production of the antigen-specific, species-specific monoclonal antibodies which relies upon the in vitro conversion of blood-borne immune cells, such as macrophages and lymphocytes. Vaccine efficacy is enhanced by the administration of compositions containing component-specific immunostimulating agents and other elements, such as antigens or carrier particles, such as colloidal methods, such as gold.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/065,155, filed Nov. 10, 1997, and to U.S. Provisional Application No.60/075,811, filed Feb. 24, 1998; and to a U.S. Provisional Application,No. Not Assigned, filed Nov. 6, 1998.

FIELD OF THE INVENTION

The present invention relates generally to immunology. Morespecifically, the invention relates to methods and compositions for theenhancement of an immune response in a human or animal. Such enhancementmay result in stimulation or suppression of the immune response. Theinvention also relates to targeted component-stimulating compositionsthat easily and efficiently present antigenic components to particularimmune cells to enhance an immune response in a human or animal. Thepresent invention further relates to the use of such methods andcompositions for the production of antigen-specific, species-specificmonoclonal antibodies and the in vitro methods for production of suchantibodies.

BACKGROUND OF THE INVENTION

The introduction of desired agents into specific target cells has been achallenge to scientists for a long time. The challenge of specifictargeting of agents is to get an adequate amount of the agent or thecorrect agent to the target cells of an organism without providing toomuch exposure of the rest of the organism. A very desired target fordelivery of specific agents is the selective control of the immunesystem. The immune system is a complex response system of the body thatinvolves many different kinds of cells that have differing activities.Activation of one portion of the immune system usually causes manydifferent responses due to unwanted activation of other related portionsof the system. Currently, there are no methods or compositions forproducing the desired response by targeting the specific components ofthe immune system.

One method that has been used with limited success is the targeting ofcells that bear a specific receptor and providing an antibody to thatreceptor that acts as a carrier for an agent. The agent could be apharmaceutical agent that is a cell stimulant or the therapeutic agentcould be a radioactive moiety that causes cell death. The problemsinherent in this techniques are the isolation of the specific receptor,the production of an antibody having selective activity for thatreceptor and no cross-reactivities with other similar epitopes, andattachment of the agent to the antibody. A problem attendant to suchlimited delivery is that the agent may never be released internally inthe targeted cell, the agent is not releasably bound to the antibody andtherefore, may not be fully active or capable of any activity once it isdelivered to the site.

The immune system is a complex interactive system of the body thatinvolves a wide variety of components, including cells, cellular factorswhich interact with stimuli from both inside the body and outside thebody. Aside from its direct action, the immune system's response is alsoinfluenced by other systems of the body including the nervous,respiratory, circulatory and digestive systems.

One of the better known aspects of the immune system is its ability torespond to foreign antigens presented by invading organisms, cellularchanges within the body, or from vaccination. Some of the first kinds ofcells that respond to such activation of the immune system arephagocytes and natural killer cells. Phagocytes include among othercells, monocytes, macrophages, and polymorphonuclear neutrophils. Thesecells generally bind to the foreign antigen, internalize it and maydestroy it. They also produce soluble molecules that mediate otherimmune responses, such as inflammatory responses. Natural killer cellscan recognize and destroy certain virally-infected embryonic and tumorcells. Other factors of the immune response include both complementpathways which are capable of responding independently to foreignantigens or acting in concert with cells or antibodies.

One of the aspects of the immune system that is important forvaccination is the specific response of the immune system to aparticular pathogen or foreign antigen. Part of the response includesthe establishment of “memory” for that foreign antigen. Upon a secondaryexposure, the memory function allows for a quicker and generally greaterresponse to the foreign antigen. Lymphocytes in concert with other cellsand factors, play a major role in both the memory function and theresponse.

Generally, it is thought that the response to antigens involves bothhumoral responses and cellular responses. Humoral immune responses aremediated by non cellular factors that are released by cells and whichmay or may not be found free in the plasma or intracellular fluids. Amajor component of a humoral response of the immune system is mediatedby antibodies produced by B lymphocytes. Cell-mediated immune responsesresult from the interactions of cells, including antigen presentingcells and B lymphocytes (B cells) and T lymphocytes (T cells).

The response is initiated by the recognition of foreign antigens byvarious kinds of cells, principally macrophages or other antigenpresenting cells. This leads to activation of lymphocytes, inparticular, the lymphocytes that specifically recognize that particularforeign antigen and results in the development of the immune response,and possibly, elimination of the foreign antigen. Overlaying the immuneresponse directed at elimination of the foreign antigen are complexinteractions that lead to helper functions, stimulator functions,suppresser functions and other responses. The power of the immunesystem's responses must be carefully controlled at multiple sites forstimulation and suppression or the response will either not occur, overrespond or not cease after elimination.

The recognition phase of response to foreign antigens consists of thebinding of foreign antigens to specific receptors on immune cells. Thesereceptors generally exist prior to antigen exposure. Recognition canalso include interaction with the antigen by macrophage-like cells or byrecognition by factors within serum or bodily fluids.

In the activation phase, lymphocytes undergo at least two major changes.They proliferate, leading to expansion of the clones of antigen-specificlymphocytes and amplification of the response, and the progeny ofantigen-stimulated lymphocytes differentiate either into effector cellsor into memory cells that survive, ready to respond to re-exposure tothe antigen. There are numerous amplification mechanisms that enhancethis response.

In the effector phase, activated lymphocytes perform the functions thatmay lead to elimination of the antigen or establishment of the vaccineresponse. Such functions include cellular responses, such as regulatory,helper, stimulator, suppressor or memory functions. Many effectorfunctions require the combined participation of cells and cellularfactors. For instance, antibodies bind to foreign antigens and enhancetheir phagocytosis by blood neutrophils and mononuclear phagocytes.Complement pathways are activated and may participate in the lysis andphagocytosis of microbes in addition to triggering other body responses,such as fever.

In the immune response to antigens, immune cells interact with eachother by direct cell to cell contact or indirect cell to cell (factormediated) communication. For example, interactions between T cells,macrophages, dendritic cells, and B cells arc necessary for an effectiveimmune response. B and T cells are activated by signals from dendriticcells or macrophages, which are antigen presenting cells (APC) thatpresent antigens and deliver activation signals to resting cells.Activated T cells help control immune responses and participate in theremoval of foreign organisms. Helper T cells cause cells to becomebetter effector cells, such as helping cytotoxic T cell precursors, todevelop into killer cells, helping B cells make antibodies, and helpingincrease functions of other cells like macrophages. Activated B cellsdivide and produce antigen specific antibodies and memory B cells. Thecells involved in the immune response also secrete cellular factors orcytokines, which enhance the functions of phagocytes, stimulateinflammatory responses and effect a variety of cells.

The reactions of these cells also involve feedback loops. Macrophagesand other mononuclear phagocytes, or APCs, actively phagocytose antigensfor presentation to B and T cells and such activity can be enhanced bylymphocytic cellular factors. Macrophages also produce cytokines that,among other activities, stimulate T cell proliferation anddifferentiation, and that recruit other inflammatory cells, especiallyneutrophils, and are responsible for many of the systemic effects ofinflammation, such as fever. One such cytokine, called interleukin-12,is especially important for the development of cell-mediated immunity.

Dendritic cells are also APCs which initiate an immune response. Thereare a number of different types of dendritic cells, including lymphoiddendritic cells and Langerhans cells of the skin. They can be foundthroughout the body and particularly in the spleen, lymph nodes,tonsils, Peyer's patches, and thymus. They are irregularly shaped cellswhich continuously extend and contract dendritic (tree-like) processes.One of their roles in the immune system is to regulate and induce B andT cell activation and differentiation. They are potent accessory cellsfor the development of cytotoxic T cells, antibody formation by B cells,and some polyclonal responses like oxidative mitogenesis. They alsostimulate T cells to release the cytokine interleukin-2.

An important arm of vaccination is the response to antigens that isprovided by B lymphocytes or B cells. B cells represent about 5 to 15%of the circulating lymphocytes. B cells produce immunoglobulins, IgG,IgM, IgA, IgD, and IgE, which may be released into body fluids, secretedwith attached proteins or be inserted into the surface membrane of the Bcell. Such immobilized immunoglobulins act as specific antigenreceptors. In responding to antigen, these immunoglobulin receptors arecrosslinked, known as capping, followed by internalization anddegradation of the immunoglobulin. Capping also occurs withglycoproteins located on the surface membrane of the B cells.

The B plasma cells produce and secrete antibody molecules that can bindforeign proteins, polysaccharides, lipids, or other chemicals in extracellular or cell-associated forms. The antibodies produced by a singleplasma cell are specific for one antigen. The secreted antibodies bindthe antigen and trigger the mechanisms that facilitate theirdestruction.

Monoclonal Antibodies

One of the most widely employed aspects of the immune responsecapabilities is the production of monoclonal antibodies. The advent ofmonoclonal antibody (Mab) technology in the mid 1970s provided avaluable new therapeutic and diagnostic tool. For the first time,researchers and clinicians had access to unlimited quantities of uniformantibodies capable of binding to a predetermined antigenic site andhaving various immunological effector functions. Currently, thetechniques for production of monoclonal antibodies is well known in theart.

These monoclonal antibodies were thought to hold great promise inmedicine and diagnostics. Unfortunately, the development of therapeuticproducts based on these proteins has been limited because of problemsthat are inherent in monoclonal antibody therapy. For example, mostmonoclonal antibodies are mouse derived and, thus, do not fix humancomplement well. They also lack other important immunoglobulinfunctional characteristics when used in humans.

The biggest drawback to the use of monoclonal antibodies is the factthat nonhuman monoclonal antibodies are immunogenic when injected into ahuman patient. After injection of a foreign antibody, the immuneresponse mounted by a patient can be quite strong. The immune responsecauses the quick elimination of the foreign antibody, essentiallyeliminating the antibody's therapeutic utility after an initialtreatment. Unfortunately, once the immune system is primed to respond toforeign antibodies, later treatments with the same or different nonhumanantibodies can be ineffective or even dangerous because ofcross-reactivity.

Mice can be readily immunized with foreign antigens to produce a broadspectrum of high affinity antibodies. However, the introduction ofmurine antibodies into humans results in the production of ahuman-anti-mouse antibody (HAMA) response due to the presentation of aforeign protein in the body. Use of murine antibodies in a patient isgenerally limited to a term of days or weeks. Longer treatment periodsmay result in anaphylaxis. Moreover, once HAMA has developed in apatient, it often prevents the future use of murine antibodies fordiagnostic or therapeutic purposes.

To overcome the problem of HAMA response, researchers have attemptedseveral approaches to modify nonhuman antibodies, to make themhuman-like. These approaches include mouse/human chimers, humanization,and primatization. Early work in making more human-like antibodies usedcombined rabbit and human antibodies. The protein subunits ofantibodies, rabbit Fab fragments and human Fc fragments, were joinedthrough protein disulfide bonds to form new, artificial proteinmolecules or chimeric antibodies.

Recombinant molecular biological techniques have been used to createchimeric antibodies. Recombinant DNA technology was used to construct agene fusion between DNA sequences encoding mouse antibody variable lightand heavy chain domains and human antibody light chain (LC) and heavychain (HC) constant domains to permit expression of chimeric antibodies.These chimeric antibodies contain a large number of nonhuman amino acidsequences and are immunogenic to humans. Patients exposed to thesechimeric antibodies produce human-anti-chimera antibodies (HACA). HACAis directed against the murine V region and can also be directed againstthe novel V-region/C-region (constant region) junctions present inrecombinant chimeric antibodies.

To overcome some of the limitations presented by the immunogenicity ofchimeric antibodies, molecular biology techniques are used to createdhumanized or reshaped antibodies. The DNA sequences encoding the antigenbinding portions or complementarity determining regions (CDRs) of murinemonoclonal antibodies are grafted, by molecular means, on the DNAsequences encoding the frameworks of human antibody heavy and lightchains. The humanized Mabs contain a larger percentage of human antibodysequences than do chimeric Mabs. The end product, which comprisesapproximately 90% human antibody and 10% mouse antibody, contains amouse binding site on an human antibody. It also contains certain aminoacid substitutions from the mouse Mab into the framework of thehumanized Mab in order to retain the correct shape, and thus, bindingaffinity for the target antigen.

In practice, simply substituting murine CDRs for human CDRs is notsufficient to generate efficacious humanized antibodies retaining thespecificity of the original murine antibody. There is an additionalrequirement for the inclusion of a small number of critical murineantibody residues in the human variable region. The identity of theseresidues depends upon the structure of both the original murine antibodyand the acceptor human antibody. It is the presence of these murineantibody residues that helps create a HACA response in the patient,leading to rapid clearance of the monoclonal antibodies and the fear ofanaphylaxis.

Another technique, called resurfacing technology, is used for humanizingmouse antibodies. Resurfacing involves replacing the mouse antibodysurface with a human antibody surface in a process that is faster andmore efficient than other humanization techniques. This techniqueprovides a method of redesigning murine monoclonal antibodies toresemble human antibodies by humanizing only those amino acids that areaccessible at the surface of the V-regions of the recombinant F_(v). Theresurfacing of murine monoclonal antibodies may maintain the avidity ofthe original mouse monoclonal antibody in the reshaped version, becausethe natural framework-CDR interactions are retained. Again, theseantibodies suffer from the problem of being antigenic due to their mouseorigins.

Other technologies use primate, rather than mouse, sequences to humanizeMabs. The rationale of this approach, called primatization, is that mostof the sequences in the primate antibody variable region areindistinguishable from human sequences. Primatized anti-CD4 Mabs for thetreatment of rhumatoid arthritis and severe asthma are being developed.However, these Mabs are still foreign proteins to the immune system ofthe patient and evoke an immune response.

In an effort to avoid the immune response to foreign proteins, a varietyof approaches are being developed to make human Mabs that contain onlyhuman antibody components. One approach is to isolate a human B cellclone that naturally makes antibody to the desired antigen and grow itin a trioma cell culture system. Because human antibodies are made onlyagainst antigens that are foreign to the host, none of the human B cellswill make antibodies against human antigens. Therefore, this approach isnot useful to produce Mabs against antigens that are human proteins.

Two other approaches to create human Mabs are phage display and use oftransgenic mice. Phage display technique takes advantage of the abilityof humans to make antibodies against any possible structure. Thistechnique uses the antibody genes from many individual humans to createa large library of phage antibodies, each displaying a functionalantibody variable domain on its surface. From this library, individualvariable domains are selected for their ability to bind to the desiredantigen. The Mab is created through molecular biology techniques bycombining an antibody variable domain having the desired bindingcharacteristics and a constant domain that best meets the potentialhuman therapeutic product. Again, this technique lacks antigenspecificity. The phage library cannot contain every binding region forany and all desired antigens. It also may contain binding regions whichlack specificity. Thus, this technique may require considerableengineering to increase antibody affinities to useful levels.

Transgenic mice are also being used to create “human” antibodies. Thetransgenic mice are created by replacing mouse immunoglobulin gene lociwith human immunoglobulin loci. This approach may provide advantagesover phage display technologies because it takes advantages of mouse invivo affinity maturation machinery.

All of the current technologies for producing human or human-like Mabsare insufficient to provide a species specific antibody that is antigenspecific for a described antigen. Chimeric antibodies have theadvantages of retaining the specificity of the murine antibody andstimulating human Fc dependent complement fixation and cell-mediatedcytotoxicity. However, the murine variable regions of these chimericantibodies can still elicit a HAMA response, thereby limiting the valueof chimeric antibodies as diagnostic and therapeutic agents.

Efforts to immortalize human B-cells or to generate human hybridomascapable of producing immunoglobulins against a desired antigen have beengenerally unsuccessful, particularly with human antigens. Additionally,immune tolerance in humans prevents the successful generation ofantibodies to self-antigens.

Vaccine Therapy

Vaccines may be directed at any foreign antigen, whether from anotherorganism, a changed cell, or induced foreign attributes in a normal“self” cell. The route of administration of the foreign antigen can helpdetermine the type of immune response generated. For example, deliveryof antigens to mucosal surfaces, such as oral inoculation with livepolio virus, stimulates the immune system to produce an immune responseat the mucosal surface. Injection of antigen into muscle tissue oftenpromotes the production of a long lasting IgG response.

Vaccines may be generally divided into two types, whole and subunitvaccines. Whole vaccines may be produced from viruses or microorganismswhich have been inactivated or attenuated or have been killed. Liveattenuated vaccines have the advantage of mimicking the naturalinfection enough to trigger an immune response similar to the responseto the wild-type organism. Such vaccines generally provide a high levelof protection, especially if administered by a natural route, and somemay only require one dose to confer immunity. Another advantage of someattenuated vaccines is that they provide person-to-person passage amongmembers of the population. These advantages, however, are balanced withseveral disadvantages. Some attenuated vaccines have a limitedshelf-life and cannot withstand storage in tropical environments. Thereis also a possibility that the vaccine will revert to the virulentwild-type of the organism, causing harmful, even life-threatening,illness. The use of attenuated vaccines is contraindicated inimmunodeficient states, such as AIDS, and in pregnancy.

Killed vaccines are safer in that they cannot revert to virulence. Theyare generally more stable during transport and storage and areacceptable for use in immunocompromised patients. However, they are lesseffective than the live attenuated vaccines, usually requiring more thanone dose. Additionally, they do not provide for person-to-person passageamong members of the population.

Production of subunit vaccines require knowledge about the epitopes ofthe microorganism or cells to which the vaccine should be directed.Other considerations in designing subunit vaccines are the size of thesubunit and how well the subunit represents all of the strains of themicroorganism or cell. The current focus for development of bacterialvaccines has shifted to the generation of subunit vaccines because ofthe problems encountered in producing whole bacterial vaccines and theside effects associated with their use. Such vaccines include a typhoidvaccine based upon the Vi capsular polysaccharide and the Hib vaccine toHaemophilus influenzae.

Other vaccines which have been developed include combination vaccinesand DNA vaccines. An example of a combination vaccine is the Bordetellapertussis toxin and its surface fimbrial hemaglutinin. In DNAvaccination, the patient is administered nucleic acids encoding aprotein antigen that is then transcribed, translated and expressed insome form to produce strong, long-lived humoral and cell-mediated immuneresponses to the antigen. The nucleic acids may be administered usingviral vectors or other vectors, such as liposomes.

The immune response created by vaccines can be non-specifically enhancedby the use of adjuvants. These are a heterogeneous group of compounds orcarrier components, such as liposomes, emulsions or microspheres, withseveral different mechanisms of action.

In addition to the typical use of vaccines for protection againstdisease, vaccination is being used to fight cancer. The idea ofnon-specifically stimulating the immune system to reject tumors isnearly a century old. Coley, an early researcher in the field, usedbacterial filtrates with considerable success. Attempts to vaccinateagainst cancer with purified cytokines and immunostimulants have hadonly limited success and have been effective for only a few types oftumors.

Many diseases, in addition to cancer, are mediated by the immune system.The diseases include allergies, eczema, rhinitis, urticaria,anaphylaxis, transplant rejection, such as kidney, heart, pancreas,lung, bone, and liver transplants; rheumatic diseases, systemic lupuserthematosus, rheumatoid arthritis, seronegative spondylarthritides,sjogren's syndrome, systemic sclerosis, polymyositis, dermatomyositis,type 1 diabetes mellitus, acquired immune deficiency syndrome,Hashimoto's thyroiditis, Graves' disease, Addison's disease,polyendocrine autoimmune disease, hepatitis, sclerosing cholangitis,primary biliary cirrhosis, pernicious anemia, cocliac disease,antibody-mediated nephritis,, glomerulonephritis, Wegener'sgranulomatosis, microscopic polyarteritis, polyarteritis nodosa,pemphigus, dermatitis herpetiformis, psoriasis, vitiligo, multiplesclerosis, encephalomyelitis, Guillain-Barre syndrome, myastheniagravis, Lambert-Eaton syndrome, sclera, episclera, uveitis, chronicmucocutaneous candidiasis, Bruton's syndrome, transienthypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM syndrome,Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune hemolyticanemia, autoimmune thrombocytopenia, autoimmune neutropenia,Waldenstrom's macroglobulinemia, amyloidosis, chronic lymphocyticleukemia, and non-Hodgkin's lymphoma.

Because of the safety concerns associated with the use of attenuatedvaccines and the low efficacy of killed vaccines, there is a need in theart for compositions and methods that enhance vaccine efficacy. There isalso a need in the art for compositions and methods of enhancing theimmune system which stimulate both humoral and cell-mediated responses.There is a further need in the art for the selective adjustment of animmune response and manipulating the various components of the immunesystem to produce a desired response. Additionally, there is a need formethods and compositions that can accelerate and expand the immuneresponse for a more rapid response in activation. There is an increasedneed for the ability to vaccinate populations, of both humans andanimals, with vaccines that provide protection with just one dose.

What is needed are compositions and methods for target specific deliveryof agents to only the target cells. It would be preferable for someadministrations and treatments if the agent is internalized by thetargeted cells. Once inside the cell, the agent should be sufficientlyreleased from the transport system such that the agent is active. Suchcompositions and methods should be able to deliver therapeutic agents tothe target cells efficiently. What is also needed are compositions andmethods that can be used both in in vitro and in vivo systems.

There is also a general need for compositions of antigen specific,species specific antibodies and improved methods for producing them.There is a particular need for methods for producing completely humanantibodies having affinity for a predetermined antigen. These humanimmunoglobulins should be easily and economically produced in a mannersuitable for therapeutic and diagnostic formulation.

SUMMARY OF THE INVENTION

The present invention comprises methods and compositions for targeteddelivery of component-specific immunostimulating molecules to individualimmune cells. These component-specific immunostimulating molecules bindand stimulate specific immune cells because of specific receptors on thecells. Thus, in a mixture of different cell types, thecomponent-specific immunostimulating molecules are bound only by cellshaving the selected receptor, and cells lacking the receptor areunaffected. In some populations of immune cells, only one cell typecontains receptors that bind a given component-specificimmunostimulating agent. In other cell populations, multiple immunecells may contain the receptor that binds the component-specificimmunostimulating agent. It is also possible for a given cell type tocontain receptors for multiple component-specific immunostimulatingagents.

The methods of targeted delivery to the immune cells may be such methodsas those used for in vitro techniques such as addition to cellularcultures or media or those used for in vivo administration. In vivoadministration may include direct application to the cells or suchroutes of administration as used for delivery of vaccines to humans,animals, or other organisms.

In one embodiment, the present invention comprises methods andcompositions for the simultaneous and/or sequential targeted stimulationof specific individual components of the immune system by a putativeantigen or vaccine molecule to enhance, alter, or suppress the immuneresponse to the antigen/vaccine. Another aspect of the inventionprovides for increasing the efficacy with which antigens and vaccinesinduce an immune response. In one embodiment, the methods andcompositions of the invention are capable of the simultaneousstimulation of many different individual immune components through thepresentation of specific component-stimulating compositions.

The present invention also comprises compositions and methods for thesequential stimulation of the immune system by providingcomponent-stimulating compositions at one or more steps in an immuneresponse cascade of interacting factors and cells. In one disclosedembodiment, the specific immune components stimulated are macrophages,dendritic cells, B cells, and T cells.

In a preferred embodiment, the methods comprise the sequentialadministration of component-stimulating compositions. The compositionsmay comprise the same component-specific immunostimulating agent givenat different times or by different methods of administration, such asorally the first time and by injection the second time. In anotherpreferred embodiment, the methods comprise the sequential administrationof different component-specific immunostimulating agents. For example, afirst component-specific immunostimulating agent will stimulate aninitiating step of the immune response, followed by a lateradministration of a second component-specific immunostimulating agent tostimulate a later step of the immune response. The present inventioncontemplates administration of multiple component-specificimmunostimulating agents to initiate several pathways of the immunesystem, followed by later administrations of the same or othercomponent-specific immunostimulating agents to continue and enhance theimmune response.

Additionally, it is contemplated in the present invention that thecompositions and methods described herein can be used for stimulation ofan immune response or the suppression of an immune response.Administration of component-specific immunostimulating agents for thesuppression of immune responses can be used to control autoimmunediseases or organ rejection.

In another embodiment, the present invention comprises methods andcompositions for the production of antigen-specific, species-specificmonoclonal antibodies. These methods and compositions rely upon theconversion of immune cells. In a preferred embodiment, the methods andcompositions comprise the in vitro conversion of circulating immunecells. These cells mount a primary response to the antigen, resulting inthe production of antigen specific antibody. These selected primaryclones are then immortalized to produce cells that secrete antibodiescomprised entirely of protein from the selected species.

In a preferred embodiment of the invention the antibodies produced arewholly human monoclonal antibodies which are produced through the invitro culturing of human peripheral blood lymphocytes. A key element tothis invention is the antigenic recognition of “self” molecules. Suchself molecules include those molecules that are native or naturallyoccurring in an individual, as well as any molecule having a structurewhich is the same as that which occurs naturally in a particularspecies. This recognition reduces immunogenicity because the antibodiescontain protein from only one species.

These antibodies, however, may still result in some immunogenicitybecause the protein contained within the antibody, while from the samespecies, is foreign to the individual. In another preferred embodiment,the monoclonal antibodies produced in vitro are made from the blood of ahuman or animal and then injected into that same individual. In suchsituations, the antibodies produce little or no immunogenicity becausethe antibodies are comprised entirely of protein from that individual.

Once these primary cultures have converted to the production of antigenspecific antibody, they are immortalized, for example, by fusing withhuman immortalized cancer cells or by transfecting the antibodyproducing cells with oncogenes, such as ras, or with viruses, such asEpstein Barr virus. The resultant hybridomas are screened for specificantibody secretion and then processed, for example, by limiting dilutionprocedures to isolate a single monoclonal antibody producing cell. Theresultant human monoclonal antibody contains only human protein. Noanimal protein enters into the construction of the human monoclonalantibody. The absence of all animal protein ensures that nohuman-anti-animal antibody will result from the therapeuticadministration of these antibodies.

The methods and compositions of the present invention provide a noveland versatile approach to systems for the targeted stimulation of animmune response. In one disclosed embodiment, the present inventioncomprises component-stimulating compositions. In a preferred embodiment,the component-stimulating compositions comprise component-specificimmunostimulating agents. In another preferred embodiment, thecomponent-stimulating compositions comprise component-specificimmunostimulating agents in association with colloidal metal. In yetanother preferred embodiment, such compositions comprise an antigen incombination with a component-specific immunostimulating agent, and in afurther preferred embodiment an antigen and a component-specificimmunostimulating agent are bound to a colloidal metal, such ascolloidal gold, and the resulting chimeric molecule is presented to theimmune component.

In another disclosed embodiment, the component-stimulating compositionsof the invention comprise a delivery structure or platform, to which onemember of a binding group is bound, and the complementary member of thebinding group is bound to an antigen or is bound to a component-specificimmunostimulating agent. In a more preferred embodiment, one of thecomplementary members of the binding group is bound to acomponent-specific immunostimulating agent, and another of thecomplementary members of the binding group is bound to a putitiveantigen/vaccine. The binding group members may be selected from all suchknown paired binding groups including but not limited toantibody/antigen; enzyme/substrate; and streptavidin/biotin.

One embodiment of such a composition comprises a delivery structure orplatform with a member of a binding group reversibly bound to it. Apreferred embodiment of the present invention comprises colloidal goldas a platform that is capable of binding a member of a binding group towhich component-specific immunostimulating molecules and antigen/vaccinemolecules are bound to create a component-stimulating composition. In amore preferred embodiment, the binding group is streptavidin/biotin andthe component-specific immunostimulating molecule is a cytokine.Embodiments of the present invention may also comprise binding thecomponent-specific immunostimulating molecules or antigen/vaccine in aless specific method such as by using polycations.

The present invention also comprises presentation of antigen andcomponent-specific immunostimulating agents in a variety of differentcarrier combinations. For example, a preferred embodiment includesadministration of an antigen in association with component-specificimmunostimulating agents and colloidal gold in a liposome carrier.Additional combinations are colloidal gold particles studded with viralparticles which are the active vaccine candidate or are packaged tocontain DNA for a putative vaccine. The gold particle may also contain acytokine which can then be used to target the virus to specific immunecells. Such embodiments provide for an internal vaccine preparation thatslowly releases antigen to the immune system for a prolonged response.This type of vaccine is especially beneficial for one-timeadministration of vaccines. All types of carriers, including but notlimited to liposomes and microcapsules are contemplated in the presentinvention.

Therefore, it is an object of the invention to provide reliable andfacile methods for enhancing an immune response.

It is another object of the invention to provide methods for improvingvaccine efficacy.

Another object of the invention is to provide vaccines that giveeffective protection with only one dose administration.

Yet another object of the invention is to provide methods for thetargeted stimulation of individual immune components in a specificmanner.

A further object of the invention is to provide methods for thesimultaneous presentation of an antigen and a component-specificimmunostimulating agent to individual components of the immune system.

Another object of the present invention is to provide compositionscomprising component-specific immunostimulating agents that are capableof effecting a particular component of the immune system.

Still another object of the present invention is to provide methods andcompositions for suppressing the immune responses.

Another object of the present invention is to provide compositions forusing simultaneous/sequential component-specific agents to initiate animmune response to a primary cancer capable of not only enhancing theimmune response to the primary tumor but also mounting a systemic immuneresponse to residual disease.

Yet another object of the present invention is to provide compositionsfor using simultaneous/sequential component-specific agents to initiatean immune response to a primary cancer capable of not only enhancing theimmune response to the primary tumor but also mounting a systemic immuneresponse to residual disease.

A further object of the invention to increase theantigenicity/immunogenicity of a molecule.

Still another object of the invention to generate a antigen-specificspecies-specific monoclonal antibody in vitro.

Yet another object of the invention to generate a wholly humanmonoclonal antibody through the in vitro culturing of human peripheralblood lymphocytes.

A further object of the invention is to eliminate the problem ofantigen-specific species-specific induced immunity by providing atreatment for diseases with human antigen-specific antibodies.

Another object of the invention to produce a monoclonal antibody that isperson specific and thereby eliminates a foreign immune response.

Yet another object of the present invention to provide reliable andversatile methods and compositions for targeting the delivery of immuneenhancing agents to immune cells.

A further object of the present invention to provide methods andcompositions for targeted delivery of component-specificimmunostimulating agents in vitro and in vivo.

Still another object of the present invention are methods andcompositions for the targeted delivery of component-specificimmunostimulating agents to cells having a specific receptor.

Another object of the present invention is to provide methods andcompositions comprising a targeted delivery system that is capable ofbinding and delivering a putitive antigen/vaccine using acomponent-specific immunostimulating agent.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent contains at least one color photograph. Copies of thispatent with the color photographs will be provided by the Patent andTrademark Office upon request and payment of the necessary fee.

FIG. 1 illustrates the in vitro internalization of EGF/CG/IL-1β complexby macrophages.

FIG. 2 illustrates the in vitro internalization of EGF/CG/TNF-α complexby dendritic cells.

FIG. 3 illustrates the in vitro internalization of EGF/CG/IL-6 complexby B-cells.

FIG. 4 illustrates the in vitro internalization of EGF/CG/IL-2 complexby T-cells.

FIG. 5 illustrates the production of human-anti-human TNF-α antibodiesby the process of the invention.

FIG. 6a is a 200× bright field micrograph illustrating the giant cellformation induced by this long term incubation of isolated humanlymphocytes with colloidal gold/TNF-α. FIG. 6b is a 200× phase contrastmicrograph bright field monograph of the same cells.

FIG. 7 demonstrates the necessity of the colloidal gold to generate anantibody response to self proteins.

FIG. 8 illustrates that the gold stain is associated with free-floatingclusters of activated B-cells, not macrophages or dendritic cells.

FIG. 9 is a schematic drawing of a preferred embodiment of the presentinvention.

FIG. 10 is a graph showing the saturable binding kinetics of thedelivery platform with TNF-α.

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS Enhancement of anImmune Response

The present invention relates to compositions and methods for enhancingan immune response and increasing vaccine efficacy through thesimultaneous or sequential targeting of specific immune components. Moreparticularly, specific immune components including, but not limited to,antigen presenting cells (APCs), such as macrophages and dendriticcells, and lymphocytes, such as B cells and T cells, are individuallyeffected by one or more component-specific immunostimulating agents. Anespecially preferred embodiment provides for activation of the immuneresponse using a specific antigen in combination with thecomponent-specific immunostimulating agents. As used herein,component-specific immunostimulating agent means an agent, that isspecific for a component of the immune system, and that is capable ofeffecting that component, so that the component has an activity in theimmune response. The agent may be capable of effecting several differentcomponents of the immune system, and this capability may be employed inthe methods and compositions of the present invention. The agent may benaturally occurring or can be generated and manipulated throughmolecular biological techniques or protein receptor manipulation.

The activation of the component in the immune response may result in astimulation or suppression of other components of the immune response,leading to an overall stimulation or suppression of the immune response.For ease of expression, stimulation of immune components is describedherein, but it is understood that all responses of immune components arecontemplated by the term stimulation, including but not limited tostimulation, suppression, rejection and feedback activities.

The immune component that is effected may have multiple activities,leading to both suppression and stimulation or initiation or suppressionof feedback mechanisms. The present invention is not to be limited bythe examples of immunological responses detailed herein, butcontemplates component-specific effects in all aspects of the immunesystem.

The activation of each of the components of the immune system may besimultaneous, sequential, or any combination thereof. In one embodimentof a method of the present invention, multiple component-specificimmunostimulating agents are administered simultaneously. In thismethod, the immune system is simultaneously stimulated with fourseparate preparations, each containing a composition comprising acomponent-specific immunostimulating agent. Preferably, the compositioncomprises the component-specific immunostimulating agent associated withcolloidal metal. More preferably, the composition comprises thecomponent-specific immunostimulating agent associated with colloidalmetal of one sized particle or of different sized particles and anantigen. Most preferably, the composition comprises thecomponent-specific immunostimulating agent associated with colloidalmetal of one sized particle and antigen or of differently sizedparticles and antigen.

The inventors have found that they could use certain component-specificimmunostimulating agents provide a specific stimulatory, up regulation,effect on individual immune components. For example, Interleukin-1β(IL-1β) specifically stimulates macrophages, while TNF-α (Tumor NecrosisFactor alpha) and Flt-3 ligand specifically stimulate dendritic cells.Heat killed Mycobacterium butyricum and Interleukin-6 (IL-6) arespecific stimulators of B cells, and Interleukin-2 (IL-2) is a specificstimulator of T cells. Compositions comprising such component-specificimmunostimulating agents provide for specific activation of macrophages,dendritic cells, B cells and T cells, respectively. For example,macrophages are activated when a composition comprising thecomponent-specific immunostimulating agent IL-1β is administered. Apreferred composition is IL-1β in association with colloidal metal, anda most preferred composition is IL-1β in association with colloidalmetal and an antigen to provide a specific macrophage response to thatantigen.

Many elements of the immune response are necessary for an effectivevaccination. An embodiment of a method of simultaneous stimulation is toadminister four separate preparations of compositions ofcomponent-specific immunostimulating agents comprising 1) IL-1β formacrophages, 2) TNF-α and Flt-3 ligand for dendritic cells, 3) IL-6 forB cells, and 4) IL-2 for T cells. The component-specificimmunostimulating agent compositions may be administered by any routesknown to those skilled in the art, and may use the same route ordifferent routes, depending on the immune response desired.

In another embodiment of the methods and compositions of the presentinvention, the individual immune components are activated sequentially.For example, this sequential activation can be divided into two phases,the primer phase and the immunization phase. The primer phase comprisesstimulating APCs, preferably macrophages and dendritic cells, while theimmunization phase comprises stimulating lymphocytes, preferably B cellsand T cells. Within each of the two phases, activation of the individualimmune components may be simultaneous or sequential. For sequentialactivation, a preferred method of activation is activation ofmacrophages followed by dendritic cells, followed by B cells, followedby T cells. A most preferred method is a combined sequential activationwherein there is simultaneous activation of the macrophages anddendritic cells, followed by the simultaneous activation of B cells andT cells. This is an example of methods and compositions of multiplecomponent-specific immunostimulating agents to initiate several pathwaysof the immune system.

The methods and compositions of the present invention can be used toenhance the effectiveness of any type of vaccine. The present methodsenhance vaccine effectiveness by targeting specific immune componentsfor activation. Compositions comprising component-specificimmunostimulating agents in association with colloidal metal and antigenare used for increasing the contact between antigen and specific immunecomponent. Examples of diseases for which vaccines are currentlyavailable include, but are not limited to, cholera, diphtheria,Haemophilus, hepatitis A, hepatitis B, influenza, measles, meningitis,mumps, pertussis, small pox, pneumococcal pneumonia, polio, rabies,rubella, tetanus, tuberculosis, typhoid, Varicella-zoster, whoopingcough, and yellow fever.

The combination of route of administration and the packaging used todeliver the antigen to the immune system is a powerful tool in designingthe desired immune response. The present invention comprises methods andcompositions comprising various packaging methods, such as liposomes,microcapsules, or microspheres, that can provide long-term release ofimmune stimulating compositions. These packaging systems act likeinternal depots for holding antigen and slowly releasing antigen forimmune system activation. For example, a liposome may be filled with acomposition comprising an antigen and component-specificimmunostimulating agents associated with colloidal metal. Additionalcombinations are colloidal gold particles studded with viral particleswhich are the active vaccine candidate or are packaged to contain DNAfor a putative vaccine. The gold particle would also contain a cytokinewhich could then be used to target the virus to specific immune cells.Furthermore, one could create a fusion protein vaccine which targets twoor more potential vaccine candidates and generate a vaccine for two ormore applications. The particles may also include immunogens which havebeen chemically modified by the addition of polyethylene glycol whichmay release the material slowly.

The antigen/component-specific immunostimulating agent/metal complex isslowly released from the liposome and is recognized by the immune systemas foreign and the specific component to which the component-specificimmunostimulating agent is directed activates the immune system. Thecascade of immune response is activated more quickly by the presence ofthe component-specific immunostimulating agent and the immune responseis generated more quickly and more specifically.

Other methods and compositions contemplated in the present inventioninclude using antigen/component-specific immunostimulatingagent/colloidal metal complexes in which the colloidal metal particleshave different sizes. Sequential administration of component-specificimmunostimulating agents may be accomplished in a one doseadministration by use of these differently sized colloidal metalparticles. One dose would include four independent component-specificimmunostimulating agents complexed an antigen and each with adifferently sized colloidal metal particle. Thus, simultaneousadministration would provide sequential activation of the immunecomponents to yield a more effective vaccine and more protection for thepopulation. Other types of such single-dose administration withsequential activation could be provided by combinations of differentlysized colloidal metal particles and liposomes, or liposomes filled withdifferently sized colloidal metal particles.

Use of such a vaccination systems as described above are very importantin providing vaccines that can be administered in one dose. One doseadministration is important in treating animal populations such aslivestock or wild populations of animals. One dose administration isvital in treatment of populations that are resistant to healthcare suchas the poor, homeless, rural residents or persons in developingcountries that have inadequate health care. Many persons, in allcountries, do not have access to preventive types of health care, asvaccination. The reemergence of infectious diseases, such astuberculosis, has increased the demand for vaccines that can be givenonce and still provide long-lasting, effective protection. Thecompositions and methods of the present invention provide such effectiveprotection.

The methods and compositions of the present invention can also be usedto treat diseases in which an immune response occurs, by stimulating orsuppressing components that are a part of the immune response. Examplesof such diseases include, but are not limited to, Addison's disease,allergies, anaphylaxis, Bruton's syndrome, cancer, including solid andblood borne tumors, eczema, Hashimoto's thyroiditis, polymyositis,dermatomyositis, type 1 diabetes mellitus, acquired immune deficiencysyndrome, transplant rejection, such as kidney, heart, pancreas, lung,bone, and liver transplants, Graves' disease, polyendocrine autoimmunedisease, hepatitis, microscopic polyarteritis, polyarteritis nodosa,pemphigus, primary biliary cirrhosis, pernicious anemia, coeliacdisease, antibody-mediated nephritis, glomerulonephritis, rheumaticdiseases, systemic lupus erthematosus, rheumatoid arthritis,seronegative spondylarthritides, rhinitis, sjogren's syndrome, systemicsclerosis, sclerosing cholangitis, Wegener's granulomatosis, dermatitisherpetiformis, psoriasis, vitiligo, multiple sclerosis,encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis,Lambert-Eaton syndrome, sclera, episclera, uveitis, chronicmucocutaneous candidiasis, urticaria, transient hypogammaglobulinemia ofinfancy, myeloma, X-linked hyper IgM syndrome, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune hemolytic anemia, autoimmunethrombocytopenia, autoimmune neutropenia, Waldenstrom'smacroglobulinemia, amyloidosis, chronic lymphocytic leukemia, andnon-Hodgkin's lymphoma.

The present application claims priority to, and herein incorporates byreference, in their entirety U.S. Provisional Application No.60/065,155, filed Nov. 10, 1997, and U.S. Provisional Application No.60/075,811, filed Feb. 24, 1998; and U.S. Provisional Application, No.Not Assigned, filed Nov. 6, 1998.

Production of In Vitro Monoclonal Antibodies

The methods and compositions of the present invention can further beused to produce antigen-specific, species-specific monoclonal antibodiesthat enhance immune response. These antibodies are produced, forexample, by contacting in vitro an antigen, antigen presenting cells(APCs), immune cells, such as B cells, and optionally one or morecomponent-specific immunostimulating agents. Once antigen-specificantibodies are detected, the activated immune cells are immortalized,for example, by fusing with human immortalized cancer cells. Theresulting hybridomas can then be screened for specific antibodysecretion and a single monoclonal antibody producing cell may then beisolated.

The antigen, APCs, immune cells, and component-specificimmunostimulating agent may all be introduced into the in vitro cultureat the same time. Optionally, these various components may be addedsequentially in any order or combination. The antigen andcomponent-specific immunostimulating agent may be two distinctmolecules, or may be present in the form of a complex. For example, anantigen may be complexed with different cytokines, which when added in asequential fashion would stimulate specific cells in the culture in apredictable, stepwise fashion.

Cells, such as APCs and B cells may be obtained from any source,preferably from peripheral blood. Peripheral blood from any source maybe used to produce the antigen-specific species-specific antibodies ofthe invention. Although the most significant use of the presentinvention is the production of human-anti-human antibodies, it ispossible to use the process of the invention to develop antigen-specificspecies-specific antibodies for other animal species as well. For theproduction of human-anti-human antibodies, blood may be convenientlyobtained from the American Red Cross.

In a preferred embodiment, it is desirable to separate the buffy coatfrom the rest of the whole blood. There are two separate componentswithin the buffy coat that are important to the practice of the presentinvention. These are the antigen presenting cells (APCs), such asmacrophages, lymphocytes, Langerhanns cells, and dendritic cells, andthe B cells. B cells are also antigen presenting cells, but uponpresentation with antigen, they produce an antibody response. Onceseparated from the whole blood, the entire buffy coat may be used, orthe APCs and B cells may be separated and used individually. Either ofthese components may be isolated and frozen according to procedures wellknown to those of ordinary skill in the art, such as flow cytometry,magnetic cell separation, and cryopreservation, and used at a later timewithout affecting the generation of the antibodies.

Although any combination of antigen, antigen presenting cells (APCs),component-specific immunostimulating agents, and B cells can be employedin vitro in the present invention in any manner, a preferred preparationemploys the antigen bound to a colloidal metal. In this embodiment, thebuffy coat or APCs are placed in a vessel. Colloidal metal bound antigenis then added to the vessel and incubated with the buffy coat or APCs.

The antigen bound colloidal metal composition can be produced by themethod described below. The antigen bound colloidal metal may be addedto the buffy coat or APCs alone, or in the presence of adjuvants,immunogenic proteins, nucleotides, or accessorycytokine/immuostimulators which aid in the development of a Th2/B-cellresponse. Optionally, these adjuvants, immunogenic proteins,nucleotides, and accessory cytokine/immuostimulators may be bound to thecolloidal metal in a manner similar to that by which the antigen wasbound prior to incubation of the colloidal metal bound antigen with thebuffy coat or APCs.

If B cells are initially present, as when the entire buffy coat is used,their number may become depleted during incubation with the colloidalmetal bound antigen. Therefore, after incubation, additional B cellsare, optionally, added to the vessel. These B cells may be freshlyobtained, frozen, or those separated from the buffy coat of the samesample. The APCs in the vessel activate the B cells to produceantibodies in response to the specific antigen bound to the colloidalmetal.

Once in vitro seroconversion is confirmed, the cells are immortalized.The cells can be immortalized by several different methods, for example,by fusion with immortalized cancer cells to produce hybridomas or bytransfecting the antibody producing cells with oncogenes, such as ras,or with viruses, such as Epstein Barr virus. However, any method whichproduces immortalized cells is contemplated by the present invention.Nonlimiting examples of immortalized cancer cell lines which are usefulin the present invention are K6H6/B5 cells, HUNS-1 (U.S. Pat. No.4,720,459), KR-12 (U.S. Pat. No. 4,693,975), WIL2-S, WI-L2-729HF2 (U.S.Pat. No. 4,594,325), UC 729-6 (U.S. Pat. No. 4,451,570), SKO-007, cloneJ3 of SKO-007, GK5, and LTR-228 (U.S. Pat. No. 4,624,921).

Although immortaliztion of the primary clones may be accomplished in anymanner, the following is one preferred method. Immortalized cancer cellsare added directly to the vessel containing the seroconverted cells.After incubation, the cells are washed in serum free DMEM (Delbecco'sMinimum Essential Medium), PBS (Phosphate Buffered Saline), or any serumfree physiologic buffer. The cells may then be fused, for example, usinga 40% to 100% PEG solution diluted in serum free DMEM. The fused cellsmay then be washed and the pellet reconstituted in a 50% DMEM/RPMI mediacontaining 10% fetal bovine serum (FBS), 10% Origen™, the antigencocktail mentioned above and a selective media, such as the hybridomaselecting agent HAT at a final concentration of 10%. The cells areseeded into 96 well tissue culture plates in 150 μl aliquots. Toincrease the proliferation of clones, the cells may, optionally, bestimulated by the addition of the initial antigen orantigen/component-specific immunostimulating agent mixture such as thoseused in the initial immunizations.

The cells may be grown in HAT (hypozanthine, aminopterin, thymidine)containing medium for approximately two weeks. Then a nonselectivemedia, such as HT (hypozanthine, thymidine) is substituted for the HATas a selection drug. After another incubation of about two weeks, thecells are grown in a growth media, such as 50% DMEM/RPMI supplementedwith the antigen cocktail, 10% Origen™, and 10% FBS.

The samples can be tested for the presence of antigen-specificantibodies during any of the phases of growth and are preferably testedduring all phases of growth. This testing may be done by any commonimmunological procedure, such as RIA, EIA, ELIZA, RID, or Ouchterlonytest. Positive clones are then scaled-up from 96 well plates, forexample, to 6 well plates. At this point the clones can be frozen forlater use.

The activity of the clones can be tested by methods known to those inthe art, such as by generating ascites in pristine primed mice. Theascites are purified, and then the antibody is tested for its ability toneutralize bioactivity in a well characterized cell line, for example,the TNF sensitive cell line, WEHI 164. Clones which demonstrateneutralizing ability may then be scaled-up to generate larger quantitiesof purified antibody.

In another embodiment of the present invention, the buffy coat or APCsmay be incubated simultaneously with the colloidal metal bound antigenand optionally an adjuvant. This type of incubation has been found tochange the type of immunoresponse elicited from a Th1-like response, inwhich the colloidal metal antigen is associated with the APCs which mayor may not contain cellular elements, to a Th2-type response in whichthe colloidal metal bound antigen is associated with the free-floatingclusters of B cells.

Component-Stimulating Compositions

The compositions of the present invention comprise component-specificimmunostimulating agents. Such a composition may comprise onecomponent-specific immunostimulating agent or multiplecomponent-specific immunostimulating agents. In one preferredembodiment, the composition comprises component-specificimmunostimulating agents in association with colloidal metals. Morepreferably the compositions comprise component-specificimmunostimulating agents in association with colloidal metals and otherelements for specifically targeting the effect of the component-specificimmunostimulating agents, including, but not limited to, antigens,receptor molecules, nucleic acids, pharmaceuticals, chemotherapy agents,and carriers.

The compositions of the present invention may be delivered to the immunecomponents in any manner. In another preferred embodiment, an antigenand a component-specific immunostimulating agent are bound to acolloidal metal in such a manner that a single colloidal metal particleis bound to both the antigen and the immunostimulating agent. In anotherembodiment, multiple antigens and/or multiple component-specificimmunostimulating agents are bound to a single colloidal metal particle.The combinations of antigen and component-specific immunostimulatingagents, and other elements, with one or more colloidal metal particlesis contemplated by the present invention. Administration of one orseveral of these complexed metal particles is comprised within themethods of the present invention.

In another embodiment, the component-specific immunostimulatingmolecules of the present invention comprise a delivery structure orplatform. The component-specific immunostimulating molecule and/or theantigen/vaccine may be bound directly to the platform or may be bound tothe platform through members of a binding group. A preferred embodimentof the present invention comprises a colloidal metal as a platform thatis capable of binding a member of a binding group to whichcomponent-specific immunostimulating agents and putativeantigen/vaccines are bound to create a targeted immune-enhancing agent.In a most preferred embodiment, the binding group is streptavidin/biotinand the component-specific immunostimulating agent is a cytokine.Embodiments of the present invention may also comprise binding theantigen/vaccine in a less specific method, without the use of bindingpartners, such as by using polycations or proteins.

The present invention comprises methods and compositions for targeteddelivery of component-specific immunostimulating molecules that usecolloidal metals as a platform. Such colloidal metals bind, eitherreversibly or irreversibly, molecules that interact with either anantigen/vaccine or component-specific immunostimulating agents orantigen/vaccine. The interacting molecules may either be specificbinding molecules, such as members of a binding pair, or may be rathernonspecific interacting molecules that bind less specifically. Thepresent invention contemplates the use of interacting molecules such aspolycationic elements known to those skilled in the art including, butnot limited to, polylysine, protamine sulfate, histones orasialoglycoproteins.

The members of the binding pair comprise any such binding pairs known tothose skilled in the art, including but not limited to, antibody-antigenpairs, enzyme-substrate pairs; receptor-ligand pairs; andstreptavidin-biotin. Novel binding partners may be specificallydesigned. An essential element of the binding partners is the specificbinding between one of the binding pair with the other member of thebinding pair, such that the binding partners are capable of being joinedspecifically. Another desired element of the binding members is thateach member is capable of binding or being bound to either an effectormolecule or a targeting molecule.

The compositions and methods of the present invention comprise thevariations and combinations of mixtures of the above described bindingcapabilities and methods. For example, an embodiment of the presentinvention comprises the component-specific immunostimulating moleculesbound directly to the metallic platform and the antigen/vaccine beingbound to the metallic platform through either specific or less specificbinding by integrating molecules, such as binding the binding pairsdescribed above. Another embodiment of the present invention comprisesthe antigen or vaccine bound directly to the colloidal metal platformand the component-specific immunostimulating molecule bound througheither specific or less specific binding by integrating molecules. Instill another embodiment, the present invention comprises the binding ofboth the component-specific immunostimulating molecules and theantigen/vaccine to the metallic platform through specific or lessspecific binding by integrating molecules or the direct binding of thecomponent-specific immunostimulating molecules and the antigen/vaccineto the metallic platform. A still further embodiment of the presentinvention comprises binding the component-specific immunostimulatingmolecules bound to the metallic platform through binding by the lessspecific integrating molecule binding and the antigen/vaccine bound tothe metallic platform by the binding of complementary binding members.Other combinations and variations of such embodiments are contemplatedas part of the present invention.

Method for Binding of Composition Components to Platform

Each of the elements of the compositions may be bound, separately or incombinations, to the colloidal metal by any method. However, a preferredmethod for binding the elements to the colloidal metal is as follows. Inthis example, the composition comprises an antigen and acomponent-specific immunostimulating agent, though the method is notlimited to this embodiment. The antigen is reconstituted in water.Approximately 50 to 100 μg of antigen is then incubated with colloidalmetal.

The pH of the colloidal antigen mixture may have to be adjusted so thatit is 1-3 pH unites above the pI of the component specific agent.Subsequently, 50-100 μg of the component specific agent is added to theantigen colloidal mixture and incubated for an additional 24 hours.During this time, the targeting component specific agent becomesincorporated into the antigen gold complex resulting in an immunecomponent targeted antigen delivery system. The inventors havesuccessfully performed such experiments and in fact have linked up to 3different moieties on the same colloidal metal particle.

After the binding of the component specific agent to the antigen/Au themixture is stabilized by the addition of a 1% v/v solution of 1-100%polyethylene glycol. Other stabilizing agents may include Brij 58 andcysteine, other sulfhydryl containing compounds, phospholipids,polyvinylpyrolidone, poly-L-lysine and/or poly-L-proline. The mixtureis) stabilized overnight and subsequently centrifuged to separate thebound antigen and component specific agent from unbound material. Themixture is centrifuged at 14,000 rpms for 30 min., the supernatantremoved and the pellet resuspended in water containing 1% albumin. Thisprocedure has a relatively high efficiency of coupling the antigen andtargeting component since 75% to 95% of both moieties are bound.Furthermore, free material which is not bound to the colloid isseparated by centrifugation.

Exemplified Components

The term “colloidal metal,” as used herein, includes any water-insolublemetal particle or metallic compound as well as colloids of non-metalorigin such as collodial carbon dispersed in liquid or water (ahydrosol). Examples of colloidal metals which can be used in the presentinvention include, but are not limited to, metals in groups IIA, IB, IIBand IIIB of the periodic table, as well as the transition metals,especially those of group VIII. Preferred metals include gold, silver,aluminum, ruthenium, zinc, iron, nickel and calcium. Other suitablemetals may also include the following in all of their various oxidationstates: lithium, sodium, magnesium, potassium, scandium, titanium,vanadium, chromium, manganese, cobalt, copper, gallium, strontium,niobium, molybdenum, palladium, indium, tin, tungsten, rhenium,platinum, and gadolinium. The metals are preferably provided in ionicform (preferably derived from an appropriate metal compound), forexample, the Al³⁺, Ru³⁺, Zn²⁺, Fe³⁺, Ni²⁺ and Ca²⁺ ions. A preferredmetal is gold, particularly in the form of Au³⁺. An especially preferredform of colloidal gold is HAuCl₄ (E-Y Laboratories, Inc., San Mateo,Calif.). Another preferred metal is silver, particularly in a sodiumborate buffer, having the concentration of between approximately 0.1%and 0.001%, and most preferably as approximately a 0.01% solution. Thecolor of such a colloidal silver solution is yellow and the colloidalparticles range from 1 to 40 nanometers. Such metal ions may be presentin the complex alone or with other inorganic ions.

Any antigen may be used in the present invention. Examples of antigensuseful in the present invention include, but are not limited to,Interleukin-1 (“IL-1”), Interleukin-2 (“IL-2”), Interleukin-3 (“IL-3”),Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”),Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10(“IL-10”), Interleukin-11 (“IL-11”), Interleukin-12 (“IL-12”),Interleukin-13 (“IL-13”), lipid A, phospholipase A2, endotoxins,staphylococcal enterotoxin B and other toxins, Type I Interferon, TypeII Interferon, Tumor Necrosis Factor (“TNF-α”), Transforming GrowthFactor-β (“TGF-β”)Lymphotoxin, Migration Inhibition Factor,Granulocyte-Macrophage Colony-Stimulating Factor (“CSF”),Monocyte-Macrophage CSF, Granulocyte CSF, vascular epithelial growthfactor (“VEGF”), Angiogenin, transforming growth factor (“TGF-α”), heatshock proteins, carbohydrate moieties of blood groups, Rh factors,fibroblast growth factor, and other inflammatory and immune regulatoryproteins, nucleotides, DNA, RNA, mRNA, sense, antisense, cancer cellspecific antigens; such as MART, MAGE, BAGE, and heat shock proteins(HSPs); mutant p53; tyrosinase; autoimmune antigens; immunotherapydrugs, such as AZT; and angiogenic and anti-angiogenic drugs, such asangiostatin, endostatin, and basic fibroblast growth factor, andvascular endothelial growth factor (VEGF).

The component-specific immunostimulating agent may be any molecule orcompound which increases the APC's ability to stimulate the B cell'sproduction of antibodies. Examples of component-specificimmunostimulating agents include, but are not limited to, antigens,colloidal metals, adjuvants, receptor moelcules, nucleic acids,immunogenic proteins, and accessory cytokine/immuostimulators,pharmaceuticals, chemotherapy agents, and carriers. Thesecomponent-specific immunostimulating agents may be employed separately,or in combinations. They may be employed in a free state or incomplexes, such as in combination with a colloidal metal.

Adjuvants useful in the invention include, but are not limited to, heatkilled M. butyricum and M. tuberculosis. Nonlimiting examples ofnucleotides are DNA, RNA, mRNA, sense, and antisense. Examples ofimmunogenic proteins include, but are not limited to, KLH (KeyholeLimpet Cyanin), thyroglobulin, and fusion proteins which have adjuvantand antigen moieties encoded in the gene.

Accessory cytokine/immuostimulators include, but are not limited to,Interleukin-1 (“IL-1”), Interleukin-2 (“IL-2”), Interleukin-3 (“IL-3”),Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”),Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10(“IL-10”), Interleukin-11 (“IL-11”), Interleukin-12 (“IL-12”),Interleukin-13 (“IL-13”), lipid A, phospholipase A2, endotoxins,staphylococcal enterotoxin B and other toxins, Type I Interferon, TypeII Interferon, Tumor Necrosis Factor (“TNF-α”), Transforming GrowthFactor-β (“TGF-β”) Lymphotoxin, Migration Inhibition Factor,Granulocyte-Macrophage Colony-Stimulating Factor (“CSF”),Monocyte-Macrophage CSF, Granulocyte CSF, vascular epithelial growthfactor (“VEGF”), Angiogenin, transforming growth factor (“TGF-α”), heatshock proteins, carbohydrate moieties of blood groups, Rh factors,fibroblast growth factor, and other inflammatory and immune regulatoryproteins, nucleotides, DNA, RNA, mRNA, sense, antisense, cancer cellspecific antigens; such as MART, MAGE, BAGE, and HSPs; flt3ligand/receptor system; B7 family of molecules and receptors; CD 40ligand/receptor; and immunotherapy drugs, such as AZT; and angiogenicand anti-angiogenic drugs, such as angiostatin, endostatin, and basicfibroblast growth factor, and vascular endothelial growth factor (VEGF).

Methods and compositions, other than the use of colloidal metal, can beused to deliver the component-specific immunostimulating agents, aloneor in combination with antigens or other elements. For example, thecompositions may be encapsulated in a liposome or microsphere or may bedelivered by means of other cell delivery vehicles, such as a viralvector. Additional combinations are colloidal gold particles studdedwith viral particles which are the active vaccine candidate or arepackaged to contain DNA for a putative vaccine. The gold particle wouldalso contain a cytokine which could then be used to target the virus tospecific immune cells. Furthermore, one could create a fusion proteinvaccine targets two or more potential vaccine candidates and generate avaccine for two or more applications. The particles may also includeimmunogens which have been chemically modified by the addition ofpolyethylene glycol which may release the material slowly.

The component-specific immunostimulating agents may be delivered intheir nucleic acid form, using known gene therapy methods, and producetheir effect after translation. Additional elements for activation ofimmune components, such as antigens, could be delivered simultaneouslyor sequentially so that the cellularly translated component-specificimmunostimulating agents and externally added elements work in concertto specifically target the immune response.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXPERIMENTAL DATA

EXAMPLE 1

The following is the general experimental protocol that was followed forbinding a molecule, whether antigen or component-specificimmunostimulating agent, to colloidal gold. The molecule wasreconstituted in water. 200 μg of the molecule was incubated with 25 mLcolloidal gold for 24 hours. The molecule/colloidal gold complexsolution was then centrifuged at 14,000 rpm for 20 minutes in a microcentrifuge at room temperature. The supernatant was then removed fromthe pellet.

EXAMPLE 2

50 μg of epidermal growth factor (EGF) was bound to 25 ml of 40 nmcolloidal gold particles at a pH of 11.0. The solution rocked on rockingplatform for 24 hours. 50 μg (added as 50 μl) of targeting cytokine(i.e., IL-1β to target macrophages, IL-2 to target T cells, IL-6 totarget B cells, and either TNF alpha or Flt-3 Ligand to target dendriticcells) was added to the EGF/Au solution and rocked for an additional 24hours. To separate colloidal gold bound and unbound material thesolution was then centrifuged at 14,000 rpm. The supernatant was removedand the pellet was reconstituted in 1 ml of water containing 1% humanserum albumin.

EXAMPLE 3

EGF was bound to colloidal gold (CG) using the procedure in Example 2.Tumor Necrosis Factor-α (TNF-α) was then bound to the EGF/CG complexusing the procedure of Example 1 to produce an EGF/CG/TNF-α chimera.

EXAMPLE 4

EGF was bound to colloidal gold (CG) using the procedure in Example 2.Interleukin-6 (IL-6) was then bound to the EGF/CG complex using theprocedure of Example 1 to produce an EGF/CG/IL-6 chimera.

EXAMPLE 5

EGF was bound to colloidal gold (CG) using the procedure in Example 2.Interleukin-2 (IL-2) was then bound to the EGF/CG complex using theprocedure of Example 1 to produce an EGF/CG/IL-2 chimera.

EXAMPLE 6

The buffy coat was separated from a sample of whole blood as is wellknown in the art. 100-500 mL of whole blood was collected on heparin.The blood was carefully layered onto a 50% (v/v) ficoll-hypaque solutionand centrifuged at 2700 rpm for 7 minutes. The buffy coat, thecollection of white blood cells at the serum/ficoll interface, wascollected with a Pasteur pipette and placed into 10 mL of PBS containing0.5 mg/mL heparin. The were centrifuged at 1500 rpm and the pelletwashed and recentrifuged. The cells were washed 2× in the PBS solutionand centrifuged once again.

The cells were resuspended in RPMI containing either 10% fetal bovine ornormal human serum and cultured in 6-well plates at a cell density of10⁶ cells/well. The cells were then stimulated with 50-100 μL of one orall of the antigen/cytokine mixtures.

As shown in FIG. 1, only macrophages internalized the EGF/CG/IL-1βchimera, while only dendritic cells internalized the EGF/CG/TNF-αchimera (FIG. 2). Similarly, only B cells internalized the EGF/CG/IL-6chimera (FIG. 3), and only T cells internalized the EGF/CG/IL-2 chimera(FIG. 4).

As shown by this experiment, certain component-specificimmunostimulating agents are specific for individual immune components.Thus, it is possible to target specific immune components withcomponent-specific immunostimulating agents, thereby enhancing theirimmune response resulting in increased activity in the overall immuneresponse.

EXAMPLE 7

For this example staphyloccal enterotoxin B was used as the putativeantigen/vaccine molecule, since there is evidence that binding the toxinto colloidal gold reduces its toxicity. 500 μg of the toxin wasinitially bound to 250 ml of 40 nm colloidal gold particles. Thecolloidal solution was then aliquotted. 50 ug of a targeting cytokine(IL-1βB, IL-2, IL-6 and TNFα) was added to one of the aliquots andre-incubated for 24 hours. The toxin-AU-cytokine colloid was centrifugedat 14,000 rpm and the supernatant removed. The pellet was reconstitutedto 1 ml of water. The pellet was assayed for cytokine concentration byeither sandwich or competitive ELISA. This was done to determine theamount of neat cytokine (unbound) that was to be injected in controlanimals receiving saline or toxin alone.

The immunization strategy involved simultaneous or sequentialadministration of neat toxin/cytokine mixture (as composition controls)or the toxin-Au-cytokine chimera. 5 mice/group were injected on days 1,5 & 9 with either 2.5 ug neat toxin or the same dose of toxin/cytokinemixture bound to colloidal gold. During the 14 day immunization periodtwo additional groups of mice received the neat toxin/cytokinetoxin-Au-cytokine following the schedule provided in Table 1.

TABLE 1 Day Group Type Treatment Injected 1 Control Neat toxin + NeatIL-1β + Neat TNFα Gold Toxin-Au-IL-1β + Toxin-Au-TNFα 5 Control Neattoxin + Neat IL-6 Gold Toxin-AU-IL-6 9 Control Neat toxin + Neat IL-2Gold Toxin-AU-IL-2

All groups were rechallenged with 1 μg of neat toxin alone on day 30.Protective immunization was demonstrated by the reduced or lack ofability of the neat toxin to induce morbidity. The key observation isthat the toxin bound to colloidal gold greatly reduced the toxicity ofthe toxin. Secondly, serum antibody titers to the toxin were 10× higherthan those receiving neat treatment alone. However, the serum antibodiesof animals receiving the sequential treatment were 100 times greaterthan the animals receiving the neat treatment. Finally, upon therechallenge with the neat toxin 100% of the animals treated with toxindied whereas only 20% fatality was observed in the simultaneous group.

Thus the compositions and methods of the present invention can be usedto increase the efficacy of a vaccine.

EXAMPLE 8

Binding of Cytokine to Colloidal Gold

Human TNF-α was reconstituted in water at a pH of 11 to a finalconcentration of 1 ug/ml. 300 ug of recombinant human TNF-α wasincubated overnight with 25 ml of 30-40 nm colloidal gold particles on arocking platform while mixing.

The 25 ml of colloidal gold bound TNF-α solution was divided in half.One aliquot was blocked with 125 μl of 100% PEG solution. The otheraliquot was not blocked. The two aliquots were placed back on therocking platform and incubated an additional 1 to 5 days.

The two aliquots were then centrifuged at 14,000 rpm for 20 minutes. Thesupernatant was then removed from the pellet. The pellets were blockedby reconstitution with 10 ml of a 1% solution of human serum albumin(HSA) in water at a pH of 11.

The aliquots were mixed on a rocking platform for 6 hours. The aliquotswere then centrifuged at 14,000 rpm for 20 minutes and reconstituted in3.5 ml of 1% HSA in water at a pH of 11.

EXAMPLE 9

Generation of Human-Anti-Human TNF-α Antibodies The buffy coat wasseparated from peripheral blood by ficollation and washed with PBScontaining 0.5 mg/ml heparin and EDTA. The cells were cultured for twoweeks in RPMI with ten percent (10%) heat inactivated fetal bovineserum, ten percent (10%) ORIGEN™ and 100 ng/ml of cytokine cocktailwhich is composed of the TNF-α/colloidal gold complex of Example 8 alongwith the following cytokines: IL-4, IL-6, IL-7, IL-10, IL-11, stem cellfactor (“SCF”), GMCSF, and GSF, both alone and bound to colloidal gold.

EXAMPLE 10

ELISA Assay for Human-Anti-Human TNF-α Antibodies

1 ml aliquots were taken from three of the flasks of cells treated asshown in Example 9 and centrifuged at 1,500 rpm for 15 minutes. Thesupernatant was collected and stored at −20° C.

Recombinant human TNF was coated onto the wells of a microtiter plate ina carbonate/bicarbonate buffer. The plate was washed four times with TBShaving 2.0 ml/l Tween 20. 100 μl of the supernatant was added to eachwell. Control wells received unused growth medium. The samples wereincubated overnight at room temperature.

The plates were then washed and 100 μl of an alkaline-phosphataseconjugated goat-anti-human IgG, diluted 1:1000 (in TBS+0.1% BSA), wasincubated with the wells for 1 hour. The plates were then rewashed and100 μl of the alkaline phosphatase substrate (pNPP) was incubated withthe wells until appropriate color developed.

The results of this assay are illustrated in FIG. 5. This figure showsthat human-anti-human TNF-α antibodies were produced by the method ofthe invention as described in Examples 8 and 9.

EXAMPLE 11

Cell Fusion and Hybridoma Selection

Once in vitro seroconversion of the cells in Example 9 was confirmed,10⁶−10⁷ K6H6/B5 myeloma cells were added directly into the vessel inwhich the seroconverted cells were detected. The cells were gentlymixed, collected and centrifuged at 1,200 rpm for 15 minutes. Thesupernatant was removed, and the pellet washed in serum free DMEM. Thecells were centrifuged one last time, and the supernatant completelyremoved. The pellet was gently tapped loose, and the cells fused by theaddition of a 53% PEG 1450 solution according to the strategy describedin the table below.

The PEG solution was added to the cells using the following method andincubations, while shaking the cells at 37° C.:

Time Volume of PEG added (dropwise) 0 min 0.5 ml over 30 seconds andwait 30 seconds 1 min 0.5 ml over 30 seconds and wait 30 seconds 2 min1.0 ml over 60 seconds and wait 60 seconds

Next, serum free DMEM was added to the cells using the followingschedule.

Time Volume of DMEM added (dropwise) 0 min  1.0 ml over 30 seconds andwait 30 seconds 4 min  1.0 ml over 30 seconds and Wait 30 seconds 5 min 8.0 ml over 60 seconds and wait 60 seconds 7 min 15.0 ml over 60seconds and incubate 1 minute

The cells were subsequently centrifuged at 1,200 rpm for 15 minutes. Thesupernatant was removed, and the pellet was reconstituted in a 50%DMEM/RPMI media containing 10% FBS, 10% Origen™, the cytokine cocktailmentioned above and the hybridoma selecting agent HAT at a finalconcentration of 10%. The cells were initially seeded into five 96 welltissue culture clusters in 150 μl aliquots. To increase theproliferation of clones, the cells were also stimulated with 25 μlcolloidal gold bound TNF-α used in the initial immunizations.

The cells were grown in HAT containing medium for two weeks, after whichHT was substituted for the HAT as a selection drug. Following two weeksof growth, the cells were grown in 50% DMEM/RPMI media supplemented withthe cytokine cocktail, 10% Origen™, and 10% FBS.

EXAMPLE 12

Testing of Supernatants for Positive Antibody Function

The presence of TNF-α-specific antibodies in the samples in Example 13was tested during all phases of the growth. The supernatants wereinitially tested by direct EIA and then by an in vitro assay whichmeasures the inhibition of proliferation of WEHI cells in adose-dependent manner by TNF-α. Positive clones were scaled-up fromoriginal 96 well plates to 6 well plates. Subsequently, all clonestesting positive were scaled-up for cryopreservation, as well as thegeneration of 5 ml of ascites in pristine primed mice. The ascites werepurified, and the antibody tested for its ability to prevent theinhibition of proliferation of WEHI cells by TNF-α. The ability of thepurified antibody to block bioactivity indicated its neutralizingactivity.

Clones demonstrating neutralizing activity were scaled-up to generate10-100 mg of purified antibody. These antibodies were initially screenedfor the in vivo neutralization of exogenously administered TNF-α.

EXAMPLE 13

Effect of Colloidal Gold Bound TNF-α on Cell Surface Markers Determinedby Flow Cytometry

Buffy coats were obtained from the American Red Cross and were separatedusing ficol. The lymphocytes were washed 3 times with PBS containing 0.2mg/ml heparin and again treated with ficol. After washing, 1-5 millioncells per well were seeded in 9-12 well tissue culture clusters in DMEMsupplemented with 10% FBS.

Each well contained 2 ml of either (1) media alone, (2) 0.5 ug/ml of themitogen phytohemaglutinin (PHA) (for the induction of a T cellresponse), (3) 1.0 ug/ml of the mitogen lipopolysaccharides (LPS) (forthe induction of an inflammatory response), or (4) a combination of themitogens LPS & PHA each at a final concentration of 0.5 & 1.0 ug/ml,respectively. Note that it is possible to use other mitogens, such asPokeweed mitogens, as well as other agents including superantigens, suchas staphylococcal enterotoxin A and B in this assay. The cells werestimulated with either mitogens (PHA or LPS) alone, or mitogens in thepresence of either gold/TNF-α which has been stabilized withpolyethylene glycol (PEG) in HSA (human serum albumin) or gold/TNF-αwhich has been treated with HSA alone. The culture plates were harvestedfor flow cytometric analysis of the cell surface, cell activationmarkers, and cytokine expressions.

The cells were analyzed for changes in their CD4, CD8 and CD19 levels aswell as activational marker CD69 using a Beckton Dickinson Facscaliburand the Becton Dickinson tritest MAB set. This was done by collectingthe cells, centrifuging, and removing 1.8 ml of supernatant. The cellswere titurated, i.e., the cellular pellet was resuspended, and 75 μlsamples were incubated with the appropriate MAB according to themanufacturer's instructions.

Although flow cytometry did not detect any differences in the CD4, CD8or CD19 levels between the control and gold treated cells 24-48 hoursafter treatment, visual inspection of the plates revealed thatcluster-like formations had formed in both naive and gold treated cells.Yet, in the cells treated with colloidal-gold conjugated TNF-α, thenumber of clusters appeared larger and in greater frequency and celldensity. More interestingly the surrounding cells appeared to migratetoward the gold treated clusters. This may reflect the TNF-α leachingoff the gold because there was a definite gradation of cell migration.In addition, the cellular migration was not as striking in cells treatedwith PEG-stabilized gold bound TNF-α, indicating that the TNF was notleaching off the gold or leaching off to a much lower degree.

Forty-eight hours after mitogen treatment typical conditioning of themedia induced by the stimulation of the white blood cells with PHA wasobserved. However, the cells in the wells treated with gold bound TNF-αthat is stabilized with PEG or that is unstabilized exhibitedsignificantly less conditioning of the media, indicating that thegold/TNF-α was blocking the PHA-induced mitogenesis. This may indicatethat the colloidal gold blocks a Th1-like T cell response.

Although flow cytometric analysis did not reveal any significant changesin CD4, CD8, or CD19 cell populations within 24-48 hours afterstimulation, trafficking of the colloidal gold bound TNF-α into severalcell types was observed. While control cells had a normal transparentphenotype, the stabilized and unstabilized gold treated cells hadconcentrations of the gold stain in several locations on the cells aswell as cell clusters. The distribution of the gold was varied from acentral intracellular location to the cell surface. Also, the materialappeared in multiple cell types, including rounded as well as dendriticcells. In rounded cells the localization of the gold material was eitherin the nucleus or on one side of the cell surface. Although notidentified by cell surface markers, the rounded cells are thought to bedifferentiating monocytes/macrophages because of their ability to formgiant cells. (FIGS. 6a and 6 b) The colloidal gold stain disappearedwith time and, therefore, does not appear to be permanent. Thisindicates that the colloidal gold/TNF-α mixture was being metabolizedonce it entered the cell. However, the cells retained their ability touptake colloidal gold since the stain reappeared upon restimulation withcolloidal gold bound TNF-α.

FIG. 6a is a 200× bright field micrograph illustrating the giant cellformation induced by this long term incubation of isolated humanlymphocytes with colloidal gold/TNF-α. FIG. 6b is a 200× phase contrastmicrograph bright field monograph of the same cells.

EXAMPLE 14

Lymphocytes were isolated from the buffy coats of human peripheral bloodobtained from the American Red Cross. The lymphocytes were treated witheither (1) colloidal gold alone, (2) colloidal gold bound with humanserum albumin (HSA), or (3) colloidal gold bound with TNF-α. Each groupwas divided into two aliquots. One aliquot was blocked with 1% PEG, andthe other remained untreated.

The primary cell type which took up the gold or gold/HSA group was themacrophage. This was confirmed by giant cell formation. However, asillustrated by FIG. 7, the primary cell type taking up the gold in theTNF-α/gold group had the elongated form of dendritic cells.

This result is indicative of receptor mediated binding of the colloidalgold bound TNF-α. Additionally, the requirement of TNF-αfor thedifferentiation of dendritic cells suggests that the colloidal goldbound TNF-α retained its biologic activity.

EXAMPLE 15

This experiment was designed to determine the effect of adjuvantcomponents on the uptake of colloidal gold by isolated lymphocytes. Theexperiment was performed in the same manner as Examples 6 and 7, exceptthat an additional group of cells was included. These cells received 100μl of a 1.0 mg/ml suspension of heat killed Mycobacterium butyricum.This bacteria is routinely used in adjuvant preparation for antibodygeneration.

As illustrated in FIG. 8, the gold stain was no longer associated witheither the macrophage or dendritic cells, but was associated withfree-floating clusters of cells, which may be activated B-cells.Phenotypying studies are currently underway to confirm this hypothesis.

EXAMPLE 16

Streptavidin bound to colloidal gold exhibited saturable bindingkinetics. For this experiment 500 μg streptavidin was bound to 50 ml of32 nm colloidal gold for 1 hour. Subsequently, 5 ml of a stabilizingsolution (5% PEG 1450,0.1% BSA) was added to the tube and allowed to mixfor an additional 30 mm. The sol was centrifuged to remove unboundstreptavidin and washed 2 times with 5 ml of the stabilizing solution.After a final spin, the pellet was reconstituted to a volume of 5 mlwith the stabilizing solution. 1 ml aliquots were distributed tomicrofuge tubes. To these tubes increasing amounts of biotinylated humanTNF alpha were added. The biotinylated cytokine was incubated with thestreptavidin gold for 1 hour. The material was centrifuged at 10,000rpms for 10 mm. The resultant supernatant was collected and saved forTNF determinations. The pellets from each tube were washed 1 time withstabilizing solution and recentrifuged. The supernatant from this spinwas discarded. The pellet was reconstituted to 1 ml with stabilizingsolution and both the pellet and initial supernatant were assayed forTNF concentrations using our CYTELISA™ TNF kit. One can see that greaterthan 90% of the biotinylated TNF immunoreactivity was found in thepellet (FIG. 10.) indicating that the biotinylated TNF was captured bythe streptavidin bound gold.

EXAMPLE 17

This experiment was to evaluate the feasibility of the streptavidin goldcomplex as a targeted drug delivery system. In order for this occur thestreptavidin conjugated colloidal gold must bind both a biotinylatedtargeting ligand as well as a biotinylated therapeutic. To investigatethis, we performed the following experiment.

100 ml of a 32 nm colloidal gold solution was bound with a saturatingconcentration of streptavidin. After 1 hour the sol was centrifuged andwashed as described above. The colloidal gold bound streptavidin wasthen bound with sub-saturating concentrations of biotinylated cytokine.The material was vortexed and incubated for 1 hour at room temperature.Afterwards the sol was centrifuged and the pellet incubated with asolution of biotinylated polylysine. After a 1 hour incubation, the solwas re-centrifuged and washed. After a final spin and resuspension (thefinal volume of the sol was approximately 1 ml) 50 μg of theβgalactosidase reporter gene was incubated with the concentratedstreptavidin/biotinylated cytokine/polylysine chimera for 1 hour. Thematerial was centrifuged to remove unbound plasmid DNA. The finalconstruct (biotin EGF-SAP-Au-biotin polylysine-DNA) was centrifuged at14,000 rpms. The supernatant was assayed for the presence of DNA bydetermining its OD at 260 nm. We observed a decrease in the supernatantOD @ 260 nm from 0.95 to 0.25 after the incubation of the plasmid DNAwith the biotin EGF-SAP-Au-biotin polylysine construct. The DNA wasbound by the biotin EGF-SAP-Au-biotin polylysine-DNA and was centrifugedout of the sol into the pellet. These data show that a new drug deliverysystem was developed using avidin binding to colloidal gold.Biotinylation of the targeting and delivery payload was then used as themethod for binding these molecules to the colloidal gold based drug/genedelivery system.

It should be understood, of course, that the foregoing relates only topreferred embodiments of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

We claim:
 1. An antigen-specific, human-specific monoclonal antibodycomprised entirely of human protein, produced by a process consistingessentially of a) stimulating immune cells from a human in vitro toactivate the cells to produce a primary response to an antigen; whereinthe antigen is bond to colloidal metal and b) immortalizing theactivated immune cells; and c) selecting a monoclonal antibody producingcell.
 2. The composition of claim 1, wherein said antigen is selectedfrom the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-11, IL-12, IL-13, lipid A, phospholipase A2, endotoxins,staphylococcal enterotoxin B and other toxins, Type I interferon, TypeII interferon, TNF-α, TGF-β, lymphotoxin, migration inhibition factor,CSF, monocyte-macrophage CSF, granulocyte CSF, VEGF, angiogenin, heatshock proteins, carbohydrate moieties of blood groups, Rh factors,fibroblast growth factor, inflammatory and immune regulatory proteins,nucleotides, DNA, RNA, mRNA, sense, antisense, polynucleotides, cancercell specific antigens, mutant p53, tyrosinase, autoimmune antigens,immunotherapy drugs, and angiogenic and anti-angiogenic drugs.
 3. Amethod for the in vitro stimulation of immune cells to produceantigen-specific, species-specific monoclonal antibodies comprising a)stimulating immune cells from a human or animal in vitro to activate thecells to produce a primary response to an antigen, wherein the antigenis bond to colloidal metal; b) immortalizing the activated immune cells;and c) selecting a monoclonal antibody producing cell.
 4. The method ofclaim 3 wherein the stimulated immune cells produced in step (a) arestored and used at a later time.
 5. The method of claim 3 wherein thespecies is mammalian.
 6. The method of claim 5 wherein the species ishuman.
 7. The method of claim 3 wherein said antigen is selected fromthe group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-10, IL-11, IL-12, IL-13, lipid A, phospholipase A2, endotoxins,staphylococcal enterotoxin B and other toxins, Type I interferon, TypeII interferon, TNF-α, TGF-β, lymphotoxin, migration inhibition factor,CSF, monocyte-macrophage CSF, granulocyte CSF, VEGF, angiogenin, heatshock proteins, carbohydrate moieties of blood groups, Rh factors,fibroblast growth factor, inflammatory and immune regulatory proteins,nucleotides, DNA, RNA, mRNA, sense, antisense, polynucleotides, cancercell specific antigens, mutant p53, tyrosinase, autoimmune antigens,immunotherapy drugs, and angiogenic and anti-angiogenic drugs.
 8. Amethod for the in vitro production of antigen-specific, species-specificmonoclonal antibodies comprising a) incubating an antigen and antigenpresenting cells in vitro to activate the antigen presenting cells,wherein the antigen is bond to colloidal metal; b) adding B cells to theactivated APCs to produce primary clones; c) immortalizing the primaryclones; and d) selecting a monoclonal antibody producing cell.
 9. Themethod of claim 8 wherein the stimulated immune cells produced in step(a) are stored and used at a later time.
 10. The method of claim 8wherein steps a) and b) are performed in a single step.
 11. The methodof claim 8 wherein the activation comprises at least oneimmunostimulating molecule.
 12. The method of claim 8 wherein saidantigen is selected from the group consisting of IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, lipid A,phospholipase A2, endotoxins, staphylococcal enterotoxin B and othertoxins, Type I interferon, Type II interferon, TNF-α, TGF-β,lymphotoxin, migration inhibition factor, CSF, monocyte-macrophage CSF,granulocyte CSF, VEGF, angiogenin, heat shock proteins, carbohydratemoieties of blood groups, Rh factors, fibroblast growth factor,inflammatory and immune regulatory proteins, nucleotides, DNA, RNA,mRNA, sense, antisense, polynucleotides, cancer cell specific antigens,mutant p53, tyrosinase, autoimmune antigens, immunotherapy drugs, andangiogenic and anti-angiogenic drugs.